Lateral flow assays dominate the non-glucose rapid testing market in humans, and also other areas of application that require rapid generation of a test result, including veterinary diagnostics, agricultural testing, bio-warfare testing and food safety testing, as examples.
The advantages of the lateral flow assay system (LFIA) are well known (see Table 1). Critical among these advantages, is that they represent an appropriate point of care and field use technology that can be brought to market quickly and for a relatively small investment, and be applied over a very broad range of applications.
TABLE 1Benefits of Lateral Flow Assays in Point of Need ApplicationsKnown and mature technologyRelative ease of manufacture - equipment and processes already developedand availableEasily scalable to high volume productionStable - shelf lives of 12-24 months often without refrigerationEase of use: Minimal operator dependent steps and interpretationCan handle small volumes of multiple sample typesCan be integrated with onboard electronics, reader systems and informa-tion systemsCan have high sensitivity, specificity, good stabilityRelatively low cost and short timeline for development and approvalMarket presence and acceptance - minimal education required for usersand regulators
Traditionally designed lateral flow assays suffer from performance limitations, most notably low sensitivity and poor reproducibility. Additionally, standard format lateral flow assays produce results in the form of the presence or absence of lines at test and control regions, which have to be interpreted by eye. This subjective interpretation can often be difficult and can lead to incorrect results. These limitations have been exacerbated by the continuing use of traditional development and manufacturing practices, materials, labels, and visual detection systems. The reliance on the generation of linear features such as continuous lines to indicate the presence or absence of analyte in the system is a function of a variety of mechanical factors that can inhibit the reproducible formation of other features on the same substrate. In many instances it is desirable to test for multiple reactions in a single sample (multiplexing). The use of linear features makes multiplexing difficult, makes interpretation of competitive assay results counter-intuitive, and adds to result variability through user error.
Further, quantification and objective read/record technology, often linked to laboratory information systems (LIS) are being implemented at an increasing rate. Demand for multiplexed systems, where detection of more than one analyte is necessary in the same test system is also developing. Current Lateral Flow test systems are generally incompatible with these needs.
The limitations of the current lateral flow assay devices and methods have substantially restricted their application to relatively low specification testing. For more demanding systems requiring high sensitivity, quantification and multiplexing, and for numerous market segments such as military, consumer, environmental or veterinary testing where intuitive, fast, easily interpreted results are required, the current Lateral Flow system is inadequate.
Therefore, there is a need for devices and methods that overcome the limitations of the current technologies and methodologies, that are less subject to interpretation errors, that can produce quantitative results, that are reproducible, that can be multiplexed and that can be applied in numerous market segments. The present invention addresses these and other related needs.
The invention disclosed herein provides for such a device, and discloses methods including but not limited to, methods for manufacturing rapid assays that can produce easily interpreted results in the form of unique indicia, which indicia being representative of the results of the test. The indicia may be standardized symbols visible to the eye or a reader or may be encrypted indicia interpretable by a specialized reader device. The indicia can be developed in any orientation relative to the direction of flow of the assay. The indicia may also indicate qualitative outcomes (positive or negative), semi-quantitative or quantitative outcomes depending upon the assay and reagent design.
Lateral Flow Assay Formats
FIG. 1 shows a typical configuration for such a lateral flow assay. Traditionally designed assays are composed of a variety of materials, each serving one or more purposes, overlapping onto one another, mounted on a backing card using a pressure sensitive adhesive.
The test device consists of several zones, typically constituted by individual segments of different materials, each of which will be briefly explained here. When a test is run, a sample of the material to be tested (sample) is added to the proximal end of the strip, onto a Sample Application Pad. Here, the sample is treated by means of added predetermined reagents to make it compatible with the rest of the test. Liquid phase elements of the treated sample (which may be dissolved, suspended, emulsified or any other liquidized formats) migrate to a next segment of the test device, the Conjugate Pad. Here, a detector reagent has been immobilized, typically consisting of a protein linked passively or covalently to a signal molecule or particle, typically a colloidal gold, or a colored, fluorescent or paramagnetic monodisperse latex particle. The signal reagent can also be another reagent, including non-particulates (e.g., soluble, directly labeled fluorophores gels). This label has been conjugated to one of the specific biological components of the assay, either an antigen or an antibody, depending on the assay format of the specific test device. The liquid phase sample re-mobilizes the dried conjugate material causing it to incorporate into the liquid phase sample material, and analyte in the sample interacts with the conjugate as both migrate into the next section of the test strip, the Reaction Matrix. The reaction matrix is typically a porous membrane with a hydrophilic, open structure for the purposes of transporting liquids to the reagent and control, onto which the other specific biological components of the assay have been immobilized. These are typically proteins, either antibody or antigen, which have been laid down in bands or stripes in specific areas of the membrane where they serve to capture the components of the liquid phase sample, the analyte and conjugate, as they migrate past, through or over the capture lines. Excess liquid phase materials (sample and reagents) continue to migrate across the strip, past the capture lines and are entrapped in a Wick or absorbent pad. Test results are developed on the reaction matrix and are represented as the presence of absence of indicia (typically continuous lines) of captured conjugate which are read either by eye or using a reader device.
Assay formats are often either sandwich (direct) or competitive (competitive inhibition) in nature, and can accommodate qualitative, semi-quantitative, or in certain specific cases, fully quantitative assays.
Direct assay formats are typically used when testing for larger analytes with multiple antigenic sites, such as hCG, Dengue antibody or antigen, or HIV. In this case, a positive result is indicated by the presence of a test line. Some of the conjugated particles will not be captured at the capture line, and will continue to flow toward the second line of immobilized antibodies, the control line. This control line typically comprises a species-specific anti-immunoglobulin antibody, specific for the conjugate antibody on the conjugate.
Competitive assay formats are typically used when performing a test for small molecules with single antigenic determinants, which cannot bind to two antibodies simultaneously. In this format, a positive result is indicated by the absence of a test line on the reaction matrix. A control line should still form, irrespective of the result on the test line. The two formats are illustrated schematically in FIGS. 2a and 2b. 
Fluid Transport and Signal Development in Lateral Flow Systems
The function of the current lateral flow test device is based on capillary flow of liquids along the length of the test strip, flowing from the sample introduction pad to the absorbent pad as shown in FIG. 1. Hence the flow geometry and capillary driving force is essentially one dimensional through the reaction matrix, and through the test and control lines. Nitrocellulose membranes are the predominantly used reaction matrix in lateral flow tests. In a lateral flow device the test and control lines are typically made up of proteins but can be other types of biomarker that are bound to the Reaction Matrix in line formats, generally oriented perpendicular to the direction of flow.
An example describing the processing and use of nitrocellulose as the reaction matrix will illustrate the issues with this line format.
Purpose:
The purpose of the reaction matrix in a lateral flow assay is to bind proteins or other capture reagents at the test and control areas, and to maintain their stability and activity over the shelf life of the product. When the test is run, this matrix must accept the conjugate and sample from the conjugate pad, flow them consistently to the reaction area, allow the reaction at the test and control lines to happen and allow excess fluids, label and reactants to exit without binding.
Material:
The material of choice in the vast majority of lateral flow assay systems has historically been nitrocellulose. Several attempts have been made to introduce other material types into the market, including nylon and PVDF membranes, however those attempts have had limited success, apparently due to factors including cost, limited utility, the need for education regarding new chemistry and processing requirements, and inertia due to the large bank of existing experience in the use of nitrocellulose. Other matrices are in development, including plastic materials with controlled contact angles that allow the flow of reactants to occur on the surface of the matrix in a controlled manner.
Nitrocellulose, while extremely functional, may not always be an ideal matrix for an analytical membrane in LFIA's. It does have certain characteristics that make it useful, and it remains the only material that has been successfully and widely applied in this way to date. These characteristics include relatively low cost, true capillary flow characteristics, high protein binding capacity, relative ease of handling (with direct cast, or backed membranes) and a variety of available products with varying wicking rates and surfactant contents. However, the material also possesses a variety of characteristics that make it imperfect for this application. These include imperfect reproducibility of performance within and between lots, shelf life issues, flammability (primarily in unbacked membranes), variable characteristics due to environmental conditions, such as relative humidity, and being subject to breakage (if unbacked), compression and scoring during processing.
As a result of these issues with the material, developers and manufacturers spend a considerable amount of time and effort in optimizing chemistries that overcome some of the inherent material issues, and in developing manufacturing processes that guarantee adequate performance over the entire shelf life of the product. Careful control of the key processes of dispensing, dipping and drying, and attention to chemical and biological treatment of the membrane in order to prevent the introduction of additional variation into the finished product are critical to success.
Flow Characteristics:
In order to function as the reaction matrix in a lateral flow system, the material is typically hydrophilic and has consistent flow characteristics. Nitrocellulose as a base material is hydrophobic, and is made hydrophilic by the addition of rewetting agents during the membrane production process. These rewetting agents are surfactants, and the amount and type of surfactant, and the surfactant addition methods differ from manufacturer to manufacturer and also from brand to brand within a manufacturer. The amount and type of surfactant in the membrane can affect the performance of the assay initially and over time. Not every protein will be compatible with every surfactant. This is one reason for the requirement for screening of multiple membrane types during development. Nitrocellulose membranes' flow characteristics change over time, primarily due to desiccation of the membranes upon storage. Nitrocellulose membranes can be envisaged as a sponge, with the pores of the sponge being held open by water. If that water is removed, the pores collapse, disrupting the ability of the membrane to wick fluids through it. This results in changes and inconsistencies in flowrate over time. As a result, assays based on nitrocellulose can change their performance characteristics over time, as speed directly affects assay sensitivity, and extended run times can result in false positive issues. This is a major contributor to the variability in lateral flow assays.
Critical to the appropriate performance of a lateral flow system is the requirement that the system bind reactants only at the desired locations, namely the test and control lines. The protein binding capacity of a membrane, its interactions with proteins, and the kinetics of the protein binding process are parameters which will determine how one can apply a given set of proteins onto the membrane and how sensitive the resulting diagnostic test will be. Proteins bind to nitrocellulose through a combination of electrostatic, hydrogen and hydrophobic binding. Consistent and reproducible immobilization of immunologically active proteins to test and control lines in lateral flow or flow through assays is one of the key elements to the production of sensitive, reproducible assays.
Membrane Processing:
Nitrocellulose must undergo several processes before integration into the final device, those typically being deposition of test and control line proteins using quantitative dispensers, drying, typically using forced air ovens at elevated temperature, and immersion processes for blocking. To lay down the test and control line proteins, the membrane is striped with protein using either contact or non-contact dispensing systems, and is typically blocked thereafter to control and stabilize flowrates and hydration characteristics, and prevent non-specific binding. The dispensing method used for the test and control lines must be as quantitative as possible, and should not be subject to variation due to variations in the material hydration or absorption characteristics. Non-contact dispensing methods provide the best solution for quantitatively dispensing proteins onto nitrocellulose. The purpose of blocking a nitrocellulose membrane is to prevent binding of proteins and labeled conjugate to the membrane at areas other than the test and control lines, where it can be specifically bound. Blocking also serves other functions, including maintenance of hydration of membranes, modification of wicking rates and stabilization of test and control line proteins. Blocking is typically performed by immersion of the membranes in a solution containing proteins, surfactants and polymers, and is a relatively uncontrolled process. The blocking method must be carefully optimized and controlled to produce optimal performance in the final product over the entire shelf life of the product. Drying is subsequently performed typically by a combination of blocking to remove surface fluids and forced air at elevated temperatures. Again, this drying process must be carefully optimized and controlled to minimize variation in the final product.
This process results in the creation of one or more bound lines of capture reagent across the width of the reaction matrix. When the assay is run, the combination of the bound protein and subsequent formation of a sandwich when reacting with the flowing sample/conjugate increase the flow resistance within the test and control line regions. Resistance to flow can also be increased by the fact that the surfactant in the line has been to some degree driven away from that region by the dispensing process, resulting in a line across the membrane that is more hydrophobic than the areas before and after it, in terms of fluid flow through the matrix.
As a result, in a standard lateral flow configuration, the test and control features perturb the flow of fluid and analyte within the system. One of the primary reasons for the use of test and control lines that span the entire width of the device is to ensure that the perturbation is even across the width of the device and that flow in the longitudinal direction remains even and effectively one-dimensional. This prevents the formation of other more preferred test interpretation features, such as alpha-numeric symbols or quantitative indicia.
Current lateral Flow devices and their associated manufacturing processes impose various limitations on their use, accuracy and reproducibility.
1. Multiplexing is difficult.
a. There is a growing requirement in point of need diagnostics for the generation of assays that can detect more than one analyte in a single device. In a standard configuration, this means dispensing multiple lines perpendicular to the flow direction, separated by distances of 1 or more millimeters. A typical issue seen in multiplexed assays of this nature is “line bleed” where signal generated on one line can “bleed” into the next line, where the conjugate is physically restricted, resulting in the formation of background in the device, which lowers the sensitivity of the assay, and can result in false positives.
b. The dynamics of each assay in the system are different from each of the others. Lateral flow assays are extremely time sensitive assays. The reaction begins as soon as the sample and conjugate mix in the conjugate or sample pad, and continues during migration through the device to the test and control lines. The reaction at the test line occurs quickly, typically in less than 30 seconds. The flow rate of the reactants through the device can be extremely important to the performance of the assay. Flow rate through an analytical membrane, typically nitrocellulose, decreases in a non linear fashion with distance from the origin. As a result, the time taken for the first reaction to reach the first capture line in a multi line assay, and that taken for the reaction to reach the last line, can be significantly different. This has implications for the ability to generate quantitative assays in multiplexed formats.
2. Antibody selection must focus on antibodies with extremely high affinity and “on-rate” (Kon). This is due to the fact that the reaction at the test line must occur within only a few seconds. This makes antibody selection difficult, and means that laborious selection methods, such as dot blots or lateral flow formats must be used, as against more ergonomic methods such as ELISA, which may select for antibodies with different binding characteristics. This high affinity makes it impossible to evenly develop large diameter features in the direction of flow (including lines, which may show gradation of strength in the direction of flow). The use of smaller features (“pixels”) combined appropriately into larger features overcomes this issue.
3. Only a single format of result is generated (a horizontal line). The formation of letters, symbols and lines in any orientation other than perpendicular to the direction of flow in a lateral flow assay is made difficult by the dynamics of flow and conjugate binding in the strip. Two simple examples of the difficulty of generating alternative shapes in a lateral flow system are illustrated in FIGS. 3 and 4 (dot and +).
a. Dot: If binding reagent is dotted onto a membrane, reagent flow and binding characteristics of the binding reagent will result typically in one of three outcomes as shown in FIG. 3: (a) formation of a half moon shape, indicating that conjugate is bound at the leading edge of the dot and the rest of the dot shape does not fill. This indicates a combination of high affinity binding at the leading edge which impedes further flow through the dot, with the remainder of the reagent finding the path of least resistance around the dot; (b) a filled out but generally inconsistent dot, indicating a low affinity binding reagent, which is non optimal for the lateral flow format; and (c) no binding, indicating non specificity of the binding reagent or a negative sample.
b. Plus/Minus: A typical embodiment of this format is one where the control line would show up as the minus and the combined test and control would show up as a plus as shown in FIG. 4. In FIG. 4 is shown the actual development of the plus with the test line dispensed parallel to the 1 dimensional flow. In this case the end of test line closest to the flow introduction shows the highest level of development by the conjugate while the other end shows development only along the edges of the test line. In this case the conjugate does not flow up to the interior of the line due to the high internal flow resistance. The same result would be expected if test and control line positions were swapped in the assay.
4. Interpretation of lines is difficult, particularly in systems that rely on the eye of the user for interpretation. Additionally, lateral flow assays are typically on the order of 2-8 mm wide. The typical analytical membrane is nitrocellulose, which is an inhomogeneous material that is inconsistent both within and between lots. As a result, flow effects are commonly seen that lead to the generation of inconsistent lines across the width of the device. This inhomogeneity in line development can also be created by process-related factors, including poor lamination or cutting. This uneven line development leads to further interpretation issues, and can be particularly difficult for reader systems.
The dominant effect of one dimensional flow is shown schematically in FIGS. 5(a), (b) and (c), where a flow resistance in the form of a spot of dispensed protein is placed in the flow path before the test and control lines using dispensed drops of different volumes in different positions relative to the line. Development of the test line is perturbed directly in line with the placement of the protein spot. Lateral diffusion does occur in the system but can result in even development of the feature that the fluid reaches after the spot only if the distance between the two features is sufficient. The required distance is dependent on the diameter of the spot and the pore size of the membrane, and is generally of a distance that makes the formation of interpretable alpha numeric or other symbols impossible within the working dimensions of a test.